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Specificity of the thioester-containing reactive site of human C3 and its significance to complement activation.

ABSTRACT: The specificity of the thioester-containing site in three plasma proteins is regulated by elements of their protein structures other than the thioester bond itself. Human C4A and alpha 2-macroglobulin preferentially form amide linkages while human C3 primarily forms ester linkages with hydroxyl groups. We have examined the thioester in C3 and found evidence of strong preferences for certain carbohydrates, indications of selectivity for specific positions on those carbohydrates and a preference for terminal sugars in polysaccharides. A testable set of rules are derived from these findings which predict preferred attachment sites on polysaccharides. A computer model of the effect of different reactivities on activation of the alternative pathway of complement suggested that organisms might greatly alter their susceptibility to complement with small changes in carbohydrate structure. While a random selection of 20 biological particles showed no correlation between activation and C3b attachment efficiency, subsets of related organisms differing primarily in their surface polysaccharide exhibited stronger correlations. The strongest correlation occurred in a series of the yeasts (Cryptococcus neoformans) possessing capsular polysaccharides with one, two, three or four branching xylose sugars per repeating unit. These organisms exhibited capture efficiencies for metastable C3b from 12% (one-xylose strain) to 41% (four-xylose strain).

SUBMITTER: Sahu A

PROVIDER: S-EPMC1137246 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

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Project description:The thioester bond in complement components C3 and C4 and the protease inhibitor alpha2-macroglobulin have traditionally been thought of as fulfilling the dual roles of mediating covalent attachment and maintaining the native conformational states of these molecules. We previously reported that several human C3 thioester-region mutants, including variants E1012Q and C1010A, in the latter of which thioester-bond formation is precluded, display an unexpected phenotype. Despite the lack of a thioester bond in these mutants, they appear to adopt a native-like conformation as suggested by the finding that they are cleavable by the classical pathway C3 convertase, C4b2a, whereas the C3b-like C3(H2O) species is not. Subsequently, a species referred to as C3(NH3)* was described which potentially could account for the observations with the above mutants. C3(NH3)* is a transient species formed on aminolysis of native C3 that can spontaneously re-form the thioester bond. Importantly, it has a mobility on cation-exchange HPLC that is distinct from both native C3 and C3(H2O), but like the native molecule, it is cleavable by an alternative-pathway C3 convertase. In this study we showed by using cation-exchange HPLC as an additional conformational probe that C3 C1010A and E1012Q mutant proteins did not resemble C3(NH3)*. Instead they displayed a chromatographic behaviour that was indistinguishable from that of native C3. To assess the general applicability of these observations, we engineered the equivalent mutations into human C4, specifically C4 C1010A and C4 E1012Q. As expected, thioester-bond formation did not occur in either of these C4 mutants, but in contrast with the results with C3 we found no evidence for the formation of a stable native-like conformation in either C4 mutant, as assessed using cleavability by C1s as the conformational probe. A possible interpretation of our data is that the adoption of the native conformational state during biosynthesis of C3 and C4 is an energetically permissible process, even if it is not locked in via thioester-bond formation. Whereas this conformational state is stable in mature C3, it is unstable in mature C4, perhaps reflecting the additional post-translational cleavage of C4 before its secretion.

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Specificity of the thioester-containing reactive site of human C3 and its significance to complement activation.

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